Iranian Journal of Basic Medical Sciences ijbms.mums.ac.ir
Time-dependent changes of autophagy and apoptosis in lipopolysaccharide-induced rat acute lung injury Li Lin 1, 2, Lijun Zhang 1, 2, Liangzhu Yu 1, 2 , Lu Han 1, Wanli Ji 1, 2, Hui Shen 1, Zhenwu Hu 1, 2 * 1 School 2
of Basic Medicine, Hubei University of Science and Technology, Xianning 437100, P.R. China Hubei Province Key Laboratory on Cardiovascular, Cerebrovascular, and Metabolic Disorders, Hubei University of Science and Technology, Xianning 437100, P.R. China
ARTICLE INFO
ABSTRACT
Article type:
Objective(s): Abnormal lung cell death including autophagy and apoptosis is the central feature in acute lung injury (ALI). To identify the cellular mechanisms and the chronology by which different types of lung cell death are activated during lipopolysaccharide (LPS)-induced ALI, we decided to evaluate autophagy (by LC3-II and autophagosome) and apoptosis (by caspase-3) at different time points after LPS treatment in a rat model of LPS-induced ALI. Materials and Methods: Sprague-Dawley rats were randomly divided into two groups: control group and LPS group. ALI was induced by LPS intraperitoneal injection (3 mg/kg). The lung tissues were collected to measure lung injury score by histopathological evaluation, the protein expression of LC3-II and caspase-3 by Western blot, and microstructural changes by electron microscopy analysis. Results: During ALI, lung cell death exhibited modifications in the death process at different stages of ALI. At early stages (1 hr and 2 hr) of ALI, the mode of lung cell death started with autophagy in LPS group and reached a peak at 2 hr. As ALI process progressed, apoptosis was gradually increased in the lung tissues and reached its maximal level at later stages (6 hr), while autophagy was time-dependently decreased. Conclusion: These findings suggest that activated autophagy and apoptosis might play distinct roles at different stages of LPS-induced ALI. This information may enhance the understanding of lung pathophysiology at the cellular level during ALI and pulmonary infection, and thus help optimize the timing of innovating therapeutic approaches in future experiments with this model.
Original article
Article history:
Received: Jun 9, 2015 Accepted: Apr 7, 2016
Keywords:
Acute lung injury Apoptosis Autophagy Lipopolysaccharide
►Please cite this article as:
Lin L, Zhang L, Yu L , Han L, Ji W, Shen H, Hu Z. Time-dependent changes of autophagy and apoptosis in lipopolysaccharide-induced rat acute lung injury. Iran J Basic Med Sci 2016; 19:632-637.
Introduction
Acute lung injury (ALI) represents a leading cause of death in critically ill patients with a high mortality rate (35–45%) (1). Despite recent advances in ALI therapy, the efficacy of therapies has been modest. This slow progress may partially result from our poor understanding of the cellular and molecular basis of ALI. Additional experiments are needed to investigate the cellular and molecular basis of ALI. Recently, dysregulation of lung cell death has been reported during ALI (2). The modes of cell death in the lungs at least include (2): (i) type I programmed cell death (apoptosis), characterized by the formation of apoptotic bodies, cell shrinkage, DNA fragmentation, and chromatin condensation. (ii) type II programmed cell death (autophagy), characterized by the formation of autophagosomes, degradation of cytoplasmic contents, and little chromatin condensation. (iii) necrosis, characterized
by cellular swelling and membrane lysis. Among these, apoptosis was believed to be involved in lung injury because inhibition of apoptosis-associated signaling components such as the Fas/FasL system and caspase reportedly reduced the extent of lung injury in experimental animals (3, 4). However, the possible role of autophagy in ALI remains to be debated. On one hand, increased autophagy reportedly plays an important role in ischemia-reperfusion or ventilatorinduced lung injury (5, 6). On the other hand, autophagy-associated LC3-II has been found to interact with the Fas pathway to prevent epithelial cell apoptosis under hyperoxia (7). Further studies are needed to confirm the role of autophagy and apoptosis in experimental ALI models. Those studies examining whether different types of cell death are dynamically altered in the lung during ALI should provide important information regarding the functional role of different modes of lung cell death.
*Corresponding author: Zhenwu Hu. School of Basic Medicine, Hubei University of Science and Technology, Xianning, Hubei, P.R. China, 437100, email: [email protected]
Time course of autophagy/apoptosis in ALI
Therefore, this study aimed to investigate the timedependent changes of autophagy and apoptosis in the lungs of the rats injected with lipopolysaccharide (LPS) and their normal counterparts injected with normal saline. By comparing the lung injury, apoptosis, and autophagy in these animals, the specific role of autophagy and apoptosis in LPS-induced lung injury can be clarified.
Materials and Methods
Reagents and animals LPS was obtained from Sigma-Aldrich; primary antibodies against LC-3 and Caspase-3 were obtained from Abcam Corporation. Ninety-six healthy male Sprague-Dawley (SD) rats (180–200 g) were obtained from the Laboratory Animal Research Center of Tongji Medical College, Huazhong University of Science and Technology (Wuhan, China). Animals were maintained under specific pathogen-free conditions (temperature: 22 ± 2°C, relative humidity: 40%–80%), and allowed free access to standard laboratory food and water. LPS-induced ALI model Ninety-six healthy male SD rats were randomly divided into two groups: normal control group and LPS group. The LPS group was intraperitoneally injected with LPS (3 mg/kg) dissolved in 1 ml of sterile saline. The normal control group was injected with the same volume of saline. Lung tissues were collected prior to and 1, 2, 4, 6, and 8 hr after LPS (LPS-0 hr, LPS-1 hr, LPS-2 hr, LPS-4 hr, LPS-6 hr, LPS-8 hr) and normal saline injection (Ctrl-0 hr, Ctrl-1 hr, Ctrl-2 hr, Ctrl-4 hr, Ctrl-6 hr, and Ctrl-8 hr). Eight animals were used at each time point. All animal procedures were performed in accordance with National Institutes of Health Guide for the Care and Use of Laboratory Animals (8th Edition, revised 2011) and approved by the Animal Care and Use Committee of Hubei University of Science and Technology, China. Measurement of Lung injury Score For histopathological analysis, lung tissues were fixed in 4% formalin and embedded in paraffin. Subsequently, sections (5 μm thick) were prepared and stained with hematoxylin and eosin for pathological observation. Lung injury score of each slide was assessed by two independent pathologists in a blinded manner as previously described (8). The score represents the average of these pathologists. Each section was scored according to the following four items: (i) alveolar septal congestion, (ii) alveolar hemorrhage, (iii) intra-alveolar cell infiltrates, and (iv) intraalveolar fibrin deposition. Each item was scored from 0 to 3 according to injury field. Within each field, points were assigned according to the following criteria: alveolar septal congestion, 0: all septae are thin and delicate, 1: congested alveolar septae occur in
Iran J Basic Med Sci, Vol. 19, No. 6, Jun 2016
Lin et al
2/3 of the field; alveolar hemorrhage, 0: no hemorrhage, 1: at least 5 erythrocytes per alveolus in 1–5 alveoli, 2: at least 5 erythrocytes in 5–10 alveoli, 3: at least 5 erythrocytes in >10 alveoli; intra-alveolar fibrin deposition, 0: no intra-alveolar fibrin, 1: fibrin deposition in 2/3 of the field; intra-alveolar cell infiltrates, 0: 0–5 intra-alveolar cells per field, 1: 5–10 intra-alveolar cells per field, 2: 10–20 intra-alveolar cells per field, 3: >20 intra– alveolar cells per field. All of the points for each item were weighted according to their relative importance. The total injury score was calculated according to the following formula: total lung injury score = [(points of alveolar septal congestion/number of fields) + (points of alveolar hemorrhage/number of fields) + 2 × (points of intra-alveolar cell infiltrates/number of fields)+3 × (points of intra-alveolar fibrin deposition/ number of fields)]/total number of alveoli counted. Transmission electron microscopy Lung tissues (1 mm3) were fixed in 2.5% glutaraldehyde, followed by 1% osmium tetroxide and embedded in epoxy resin 618. Subsequently, these samples were cut into ultrathin sections and examined using a Hitachi H-300 transmission electron microscope (FEI Tecnai G2 12-type, Holland, The Netherlands). Western blot analysis Lung tissues were homogenized with a lysis buffer containing: 50 mmol/l Tris, pH 7.5, 150 mmol/l NaCl, 1% Triton X-100, 1% sodium deoxycholate, 1.0 mmol/lphenylmethanesulfonyl fluoride, 50 mmol/l sodium fluoride, 1.0 mmol/l sodium orthovanadate, 50 μg/ml pepstatin, and 50 μg/ml leupeptin. The homogenate was incubated at 4 °C for 30 min and then centrifuged for 10 min at 4 °C. The supernatant was harvested and stored at −80 °C for further analysis. Total protein content in the supernatant was measured by the BCA protein assay. Subsequently, an equal amount of proteins was separated on 15% SDS-PAGE gels and then transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA). After being blocked with 5% fat-free milk, the membranes were incubated with primary antibodies against caspase-3, cleaved caspase-3, LC3-II, or β-actin overnight at 4°C. After washing three times with TBS-T, the membranes were incubated with horseradish peroxidase (HRP) conjugated secondary antibody (Santa Cruz, USA) for 1 hr at room temperature. Protein bands were visualized using the ECL system and analyzed with a gel imaging system (Kodak system EDAS120, Tokyo, Japan). Statistical analysis All data are presented as mean±SEM. For determination of time-dependent changes in LPSinduced lung changes, the differences in all measured
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Lin et al
Figure 1. Histopathological changes and lung injury score (A) Histological structures of control and LPS groups at different time points after LPS exposure determined by H&E staining. The results showed alveolar septal widening, inflammatory infiltrates and areas of hemorrhage. (original magnifications, ×200; HE) (B) Lung injury score was determined and graded according to Matute-Bello Scoring as described in Materials and Methods. One-way ANOVA followed by a Student-Neuman-Keuls t-test was used to analyze significant differences in lung injury score at each time point between control (Ctrl, n=8 for each time point) and LPS groups (LPS, n =8 for each time point) and between different time points in control (Ctrl, n=8 for each time point) and LPS (LPS, n=8 for each time point) groups. **P
Time-dependent changes of autophagy and apoptosis in lipopolysaccharide-induced rat acute lung injury Li Lin 1, 2, Lijun Zhang 1, 2, Liangzhu Yu 1, 2 , Lu Han 1, Wanli Ji 1, 2, Hui Shen 1, Zhenwu Hu 1, 2 * 1 School 2
of Basic Medicine, Hubei University of Science and Technology, Xianning 437100, P.R. China Hubei Province Key Laboratory on Cardiovascular, Cerebrovascular, and Metabolic Disorders, Hubei University of Science and Technology, Xianning 437100, P.R. China
ARTICLE INFO
ABSTRACT
Article type:
Objective(s): Abnormal lung cell death including autophagy and apoptosis is the central feature in acute lung injury (ALI). To identify the cellular mechanisms and the chronology by which different types of lung cell death are activated during lipopolysaccharide (LPS)-induced ALI, we decided to evaluate autophagy (by LC3-II and autophagosome) and apoptosis (by caspase-3) at different time points after LPS treatment in a rat model of LPS-induced ALI. Materials and Methods: Sprague-Dawley rats were randomly divided into two groups: control group and LPS group. ALI was induced by LPS intraperitoneal injection (3 mg/kg). The lung tissues were collected to measure lung injury score by histopathological evaluation, the protein expression of LC3-II and caspase-3 by Western blot, and microstructural changes by electron microscopy analysis. Results: During ALI, lung cell death exhibited modifications in the death process at different stages of ALI. At early stages (1 hr and 2 hr) of ALI, the mode of lung cell death started with autophagy in LPS group and reached a peak at 2 hr. As ALI process progressed, apoptosis was gradually increased in the lung tissues and reached its maximal level at later stages (6 hr), while autophagy was time-dependently decreased. Conclusion: These findings suggest that activated autophagy and apoptosis might play distinct roles at different stages of LPS-induced ALI. This information may enhance the understanding of lung pathophysiology at the cellular level during ALI and pulmonary infection, and thus help optimize the timing of innovating therapeutic approaches in future experiments with this model.
Original article
Article history:
Received: Jun 9, 2015 Accepted: Apr 7, 2016
Keywords:
Acute lung injury Apoptosis Autophagy Lipopolysaccharide
►Please cite this article as:
Lin L, Zhang L, Yu L , Han L, Ji W, Shen H, Hu Z. Time-dependent changes of autophagy and apoptosis in lipopolysaccharide-induced rat acute lung injury. Iran J Basic Med Sci 2016; 19:632-637.
Introduction
Acute lung injury (ALI) represents a leading cause of death in critically ill patients with a high mortality rate (35–45%) (1). Despite recent advances in ALI therapy, the efficacy of therapies has been modest. This slow progress may partially result from our poor understanding of the cellular and molecular basis of ALI. Additional experiments are needed to investigate the cellular and molecular basis of ALI. Recently, dysregulation of lung cell death has been reported during ALI (2). The modes of cell death in the lungs at least include (2): (i) type I programmed cell death (apoptosis), characterized by the formation of apoptotic bodies, cell shrinkage, DNA fragmentation, and chromatin condensation. (ii) type II programmed cell death (autophagy), characterized by the formation of autophagosomes, degradation of cytoplasmic contents, and little chromatin condensation. (iii) necrosis, characterized
by cellular swelling and membrane lysis. Among these, apoptosis was believed to be involved in lung injury because inhibition of apoptosis-associated signaling components such as the Fas/FasL system and caspase reportedly reduced the extent of lung injury in experimental animals (3, 4). However, the possible role of autophagy in ALI remains to be debated. On one hand, increased autophagy reportedly plays an important role in ischemia-reperfusion or ventilatorinduced lung injury (5, 6). On the other hand, autophagy-associated LC3-II has been found to interact with the Fas pathway to prevent epithelial cell apoptosis under hyperoxia (7). Further studies are needed to confirm the role of autophagy and apoptosis in experimental ALI models. Those studies examining whether different types of cell death are dynamically altered in the lung during ALI should provide important information regarding the functional role of different modes of lung cell death.
*Corresponding author: Zhenwu Hu. School of Basic Medicine, Hubei University of Science and Technology, Xianning, Hubei, P.R. China, 437100, email: [email protected]
Time course of autophagy/apoptosis in ALI
Therefore, this study aimed to investigate the timedependent changes of autophagy and apoptosis in the lungs of the rats injected with lipopolysaccharide (LPS) and their normal counterparts injected with normal saline. By comparing the lung injury, apoptosis, and autophagy in these animals, the specific role of autophagy and apoptosis in LPS-induced lung injury can be clarified.
Materials and Methods
Reagents and animals LPS was obtained from Sigma-Aldrich; primary antibodies against LC-3 and Caspase-3 were obtained from Abcam Corporation. Ninety-six healthy male Sprague-Dawley (SD) rats (180–200 g) were obtained from the Laboratory Animal Research Center of Tongji Medical College, Huazhong University of Science and Technology (Wuhan, China). Animals were maintained under specific pathogen-free conditions (temperature: 22 ± 2°C, relative humidity: 40%–80%), and allowed free access to standard laboratory food and water. LPS-induced ALI model Ninety-six healthy male SD rats were randomly divided into two groups: normal control group and LPS group. The LPS group was intraperitoneally injected with LPS (3 mg/kg) dissolved in 1 ml of sterile saline. The normal control group was injected with the same volume of saline. Lung tissues were collected prior to and 1, 2, 4, 6, and 8 hr after LPS (LPS-0 hr, LPS-1 hr, LPS-2 hr, LPS-4 hr, LPS-6 hr, LPS-8 hr) and normal saline injection (Ctrl-0 hr, Ctrl-1 hr, Ctrl-2 hr, Ctrl-4 hr, Ctrl-6 hr, and Ctrl-8 hr). Eight animals were used at each time point. All animal procedures were performed in accordance with National Institutes of Health Guide for the Care and Use of Laboratory Animals (8th Edition, revised 2011) and approved by the Animal Care and Use Committee of Hubei University of Science and Technology, China. Measurement of Lung injury Score For histopathological analysis, lung tissues were fixed in 4% formalin and embedded in paraffin. Subsequently, sections (5 μm thick) were prepared and stained with hematoxylin and eosin for pathological observation. Lung injury score of each slide was assessed by two independent pathologists in a blinded manner as previously described (8). The score represents the average of these pathologists. Each section was scored according to the following four items: (i) alveolar septal congestion, (ii) alveolar hemorrhage, (iii) intra-alveolar cell infiltrates, and (iv) intraalveolar fibrin deposition. Each item was scored from 0 to 3 according to injury field. Within each field, points were assigned according to the following criteria: alveolar septal congestion, 0: all septae are thin and delicate, 1: congested alveolar septae occur in
Iran J Basic Med Sci, Vol. 19, No. 6, Jun 2016
Lin et al
2/3 of the field; alveolar hemorrhage, 0: no hemorrhage, 1: at least 5 erythrocytes per alveolus in 1–5 alveoli, 2: at least 5 erythrocytes in 5–10 alveoli, 3: at least 5 erythrocytes in >10 alveoli; intra-alveolar fibrin deposition, 0: no intra-alveolar fibrin, 1: fibrin deposition in 2/3 of the field; intra-alveolar cell infiltrates, 0: 0–5 intra-alveolar cells per field, 1: 5–10 intra-alveolar cells per field, 2: 10–20 intra-alveolar cells per field, 3: >20 intra– alveolar cells per field. All of the points for each item were weighted according to their relative importance. The total injury score was calculated according to the following formula: total lung injury score = [(points of alveolar septal congestion/number of fields) + (points of alveolar hemorrhage/number of fields) + 2 × (points of intra-alveolar cell infiltrates/number of fields)+3 × (points of intra-alveolar fibrin deposition/ number of fields)]/total number of alveoli counted. Transmission electron microscopy Lung tissues (1 mm3) were fixed in 2.5% glutaraldehyde, followed by 1% osmium tetroxide and embedded in epoxy resin 618. Subsequently, these samples were cut into ultrathin sections and examined using a Hitachi H-300 transmission electron microscope (FEI Tecnai G2 12-type, Holland, The Netherlands). Western blot analysis Lung tissues were homogenized with a lysis buffer containing: 50 mmol/l Tris, pH 7.5, 150 mmol/l NaCl, 1% Triton X-100, 1% sodium deoxycholate, 1.0 mmol/lphenylmethanesulfonyl fluoride, 50 mmol/l sodium fluoride, 1.0 mmol/l sodium orthovanadate, 50 μg/ml pepstatin, and 50 μg/ml leupeptin. The homogenate was incubated at 4 °C for 30 min and then centrifuged for 10 min at 4 °C. The supernatant was harvested and stored at −80 °C for further analysis. Total protein content in the supernatant was measured by the BCA protein assay. Subsequently, an equal amount of proteins was separated on 15% SDS-PAGE gels and then transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA). After being blocked with 5% fat-free milk, the membranes were incubated with primary antibodies against caspase-3, cleaved caspase-3, LC3-II, or β-actin overnight at 4°C. After washing three times with TBS-T, the membranes were incubated with horseradish peroxidase (HRP) conjugated secondary antibody (Santa Cruz, USA) for 1 hr at room temperature. Protein bands were visualized using the ECL system and analyzed with a gel imaging system (Kodak system EDAS120, Tokyo, Japan). Statistical analysis All data are presented as mean±SEM. For determination of time-dependent changes in LPSinduced lung changes, the differences in all measured
633
Lin et al
Figure 1. Histopathological changes and lung injury score (A) Histological structures of control and LPS groups at different time points after LPS exposure determined by H&E staining. The results showed alveolar septal widening, inflammatory infiltrates and areas of hemorrhage. (original magnifications, ×200; HE) (B) Lung injury score was determined and graded according to Matute-Bello Scoring as described in Materials and Methods. One-way ANOVA followed by a Student-Neuman-Keuls t-test was used to analyze significant differences in lung injury score at each time point between control (Ctrl, n=8 for each time point) and LPS groups (LPS, n =8 for each time point) and between different time points in control (Ctrl, n=8 for each time point) and LPS (LPS, n=8 for each time point) groups. **P
